ATP-sensitive potassium (K-ATP) channels in pancreatic beta-cells couple blood glucose levels to membrane excitability to control insulin secretion. Loss-of-function K-ATP channel mutations cause congenital hyperinsulinism, whereas gain-of-function mutations increase the risk of diabetes. Our long-term goal is to understand how regulation of K-ATP channels in pancreatic beta-cells affects insulin secretion in health and disease. To fulfill their functional role, K-ATP channels not only need to have the exquisite electrophysiological properties but also need to be delivered to the right place at the right time in order to interact with the various signaling molecules. The work conducted during the previous award period found that defective K-ATP channel trafficking that results in reduced surface channel expression is a major mechanism underlying congenital hyperinsulinism. Genetic or pharmacological manipulations that correct channel trafficking defects restore the function of some mutant channels. Our findings underscore the importance of trafficking regulation of K-ATP channels in beta-cell function. In this proposal, we aim to elucidate the trafficking pathway of K-ATP channels in beta-cells, and to study how channel trafficking is affected by disease mutations and by physiological and pharmacological stimuli. The goals of the proposed studies are: (1) to delineate the trafficking itinerary of K-ATP channels in beta-cells by determining whether channels are sorted into insulin granules prior to insertion into the plasma membrane, whether surface channels are associated with lipid rafts, and whether internalized channels are recycled; (2) to study how disease mutations affect the trafficking of K-ATP channels, and how channel trafficking defects can be corrected; (3) to investigate how physiological and pharmacological stimuli, specifically, protein kinase C activation and sulfonylurea treatments, affect K-ATP channel trafficking in beta-cells. We will use a combination of molecular, biochemical, cell biological, and electrophysiological approach to address these issues. These studies will better our understanding of the trafficking regulation of K-ATP channels in beta-cells to allow for an integrated view of the spatial and temporal control of K-ATP channel signaling, and may lead to novel therapeutic strategies for insulin secretion diseases. [unreadable] [unreadable]